Dark quencher
Encyclopedia
A dark quencher is a substance that absorbs excitation energy from a fluorophore
and dissipates the energy as heat; while a typical (fluorescent) quencher
re-emits much of this energy as light . Dark quenchers are used in molecular biology
in conjunction with fluorophores. When the two are close together, such as in a molecule or protein, the fluorophore's emission is suppressed. This effect can be used to study molecular geometry and motion.
An example of its use is in Taqman
or invader assay, SNP genotyping
methods. For instance, a hairpin loop with a fluorophore
and quencher at the base of the stem is used. An unlabeled SNP specific PCR primer (one of many) with a specific 5' tail binds to the sequence to be probed, and the taq polymerase extends the sequence that will have a specific 5' end dependent on the SNP (insensitive to polymorphisms upstream of the SNP in question). In the next run a primer
, complementary to that tail, with a hairpin loop is extended. In the next run the elongation of the complementary strand will linearise the hairpin separating the fluorophore
and quencher.
An alternative to using quenchers is to use FRET where the combination of two dyes gives a signal.
. Until the last few years, quenchers have typically been a second fluorescent dye, for example, fluorescein as the reporter and rhodamine as the quencher (FAM/TAM probes). However, quencher fluorescence can increase background noise due to overlap between the quencher and reporter fluorescence spectra. This limitation often necessitates the use of complex data analysis and optical filters. Dark quenchers offer a solution to this problem because they do not occupy an emission bandwidth. Furthermore, dark quenchers enable multiplexing (when two or more reporter-quencher probes are used together).
Fluorescent dyes absorb light, which places the dye in an excited state; the dye returns to the ground state from the excited state by emitting light (fluorescence). In a reporter – quencher system the dye nonradiatively (without light) transfers energy to the quencher. This returns the dye to the ground state
and generates the quencher excited state. The quencher then returns to the ground state through emissive decay (fluorescence
) or nonradiatively (dark quenching). In nonradiative or dark decay, energy is given off via molecular vibrations (heat). With the typical μM or less concentration of probe, the heat from radiationless decay is too small to affect the temperature of the solution.
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
and dissipates the energy as heat; while a typical (fluorescent) quencher
Quenching (fluorescence)
Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on...
re-emits much of this energy as light . Dark quenchers are used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
in conjunction with fluorophores. When the two are close together, such as in a molecule or protein, the fluorophore's emission is suppressed. This effect can be used to study molecular geometry and motion.
An example of its use is in Taqman
TaqMan
TaqMan probes are hydrolysis probes that are designed to increase the specificity of real-time PCR assays. The method was first reported in 1991 by researchers at Cetus Corporation, and the technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied...
or invader assay, SNP genotyping
SNP genotyping
SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation...
methods. For instance, a hairpin loop with a fluorophore
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
and quencher at the base of the stem is used. An unlabeled SNP specific PCR primer (one of many) with a specific 5' tail binds to the sequence to be probed, and the taq polymerase extends the sequence that will have a specific 5' end dependent on the SNP (insensitive to polymorphisms upstream of the SNP in question). In the next run a primer
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
, complementary to that tail, with a hairpin loop is extended. In the next run the elongation of the complementary strand will linearise the hairpin separating the fluorophore
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
and quencher.
An alternative to using quenchers is to use FRET where the combination of two dyes gives a signal.
Mode of function
Dark quenchers are dyes with no native fluorescenceFluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
. Until the last few years, quenchers have typically been a second fluorescent dye, for example, fluorescein as the reporter and rhodamine as the quencher (FAM/TAM probes). However, quencher fluorescence can increase background noise due to overlap between the quencher and reporter fluorescence spectra. This limitation often necessitates the use of complex data analysis and optical filters. Dark quenchers offer a solution to this problem because they do not occupy an emission bandwidth. Furthermore, dark quenchers enable multiplexing (when two or more reporter-quencher probes are used together).
Fluorescent dyes absorb light, which places the dye in an excited state; the dye returns to the ground state from the excited state by emitting light (fluorescence). In a reporter – quencher system the dye nonradiatively (without light) transfers energy to the quencher. This returns the dye to the ground state
Ground state
The ground state of a quantum mechanical system is its lowest-energy state; the energy of the ground state is known as the zero-point energy of the system. An excited state is any state with energy greater than the ground state...
and generates the quencher excited state. The quencher then returns to the ground state through emissive decay (fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
) or nonradiatively (dark quenching). In nonradiative or dark decay, energy is given off via molecular vibrations (heat). With the typical μM or less concentration of probe, the heat from radiationless decay is too small to affect the temperature of the solution.
Examples of dark quenchers
- Dabsyl (dimethylaminoazosulfonic acid) absorbs in the green spectrum and is often used with fluorescein. (Dabsyl has a nearly identical absorption, but has a sulfonyl chloride to form more stable conjugates, instead of a succinimidyl ester).
- Black Hole Quenchers are capable of quenching across the entire visible spectrum.
- Qxl quenchers span the full visible spectrum.
- Iowa black FQ absorbs in the green-yellow part of the spectrum.
- Iowa black RQ blocks in the orange-red part of the spectrum.
- IRDye QC-1 quenches dyes from the visible to the near-infrared range (500-900 nm).
Further reading
- J. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum, New York, 1999.